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Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of vanillin

A strain of Saccharomyces cerevisiae and the technology of Saccharomyces cerevisiae, which are applied in the field of genetic engineering, can solve problems such as low conversion rate, and achieve the effects of realizing industrialized production and solving the problem of utilization.

Active Publication Date: 2021-08-03
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current biotransformation is due to the instability of vanillin in microorganisms, resulting in a low conversion rate

Method used

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  • Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of vanillin
  • Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of vanillin
  • Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of vanillin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 2. Construction of strains ZR01-ZR05

[0042] The left and right 400bp sequences of the Saccharomyces cerevisiae ADH6 gene were amplified by PCR, named ADH6L, ADH6R, overlapped, and ADH6LR was obtained as donor DNA. The gRNA plasmid was constructed according to the ADH6 sequence, and the gRNA, cas9, and donor DNA were introduced into Saccharomyces cerevisiae BY4742, and the SC screening plate was spread. The obtained transformants were streaked and purified, and then the yeast genome was extracted for PCR verification and sequencing. According to the above method, ADH7, BDH1, and BDH2 were knocked out in sequence, and named ZR01.

[0043] On the basis of ZR01, ARI1 was further knocked out and named ZR02. GRE2 was knocked out on the basis of ZR01, named ZR03; HFD1 was knocked out on the basis of ZR01, named ZR04; ARI1, GRE2 and HFD1 were knocked out sequentially on the basis of ZR01, named ZR05. The primer sequences and gDNA sequences used are listed in Table 1.

[00...

Embodiment 2

[0049] The bacterial strains ZR01, ZR02, ZR03, ZR04, ZR05 constructed in Example 1 were initially added to the SD medium of 200 mg / L vanillin (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 20 g / L, completely mixed amino acid powder 2 g Bacterial strain is cultivated in SC substratum under culture medium, cultivates 48h, and its vanillin of liquid phase analysis, vanillic acid, the content of vanillyl alcohol ( figure 1 ).

[0050] The bacterial strain VAN01 constructed in Example 1 was fermented for 96h at 30°C in a YPD medium initially containing 4mmol / L ferulic acid, and the contents of ferulic acid and vanillin were analyzed in different time periods ( figure 2 ).

[0051] With the lignin hydrolyzate as the substrate, the VAN001 strain was in the YPD initially containing 40% lignin hydrolyzate for 96 hours, and its ferulic acid and vanillin contents were analyzed in different time periods ( image 3 ).

[0052] Depend on figure 1 It can be seen that the yeast st...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to saccharomyces cerevisiae engineering bacteria and application thereof in preparation of vanillin. According to the invention, on the basis of saccharomyces cerevisiae BY4742, ADH6, ADH7, BDH1 and BDH2 are knocked out, and at least one of exogenous genes 4CL and ECH is integrated; and / or at least one of endogenous genes Ari1, GRE2 and HFD1 is knocked out. According to the invention, by accurately controlling the transfer-in and knockout of the key genes, a brand new bioconversion path for synthesizing vanillin by using lignin is constructed in the yeast body, so that the metabolism path reduces the synthesis of vanillin, vanillyl alcohol and vanillic acid from vanillin, and the accumulation of vanillin is realized. Experiments show that the genetically engineered bacterium disclosed by the invention can fully utilize lignin and aromatic compounds derived from the lignin to produce vanillin in a large scale, so that not only can the problem of utilization of lignin be solved, but also industrial production of vanillin can be achieved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to engineering bacteria of Saccharomyces cerevisiae and its application in preparing vanillin. Background technique [0002] Vanillin, also known as vanillin and vanillin, has a chemical name of 4-hydroxy-3-methoxybenzaldehyde, which exists in orchid vanilla bean and is one of the most widely used aromatic compounds. It is widely used in food, beverage, perfume, and cosmetics as fragrance ingredients, and is widely used in food, medicine, cosmetics, agriculture, and intermediates in the chemical and pharmaceutical industries. Natural vanillin is mainly extracted from vanilla flower pods. Due to the need for artificial pollination in the planting of vanilla, the labor intensity is high and the work cycle is long, so the vanillin obtained by natural extraction method is far from meeting the market demand. In addition, chemical synthesis is the main way of vanillin product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/24C12N15/81C12R1/865
CPCC12N9/88C12N9/93C12N9/0006C12N9/0008C12N15/52C12N15/81C12P7/24C12Y401/02041C12Y402/01101C12Y602/01012C12Y402/03009C12Y101/01283C12Y102/01003Y02A50/30
Inventor 元英进李炳志张仁宽
Owner TIANJIN UNIV
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